Oligodendrocyte replacement and remyelination are essential to functional recovery after spinal cord injury (SCI). Therapeutic delivery of stem or progenitor cells is a promising tool as these cells produce factors that reduce inflammation, enhance survival of existing oligodendrocytes and have the capacity to differentiate into new oligodendrocytes. However, the local environment that develops after SCI does not promote differentiation into mature cells capable of functionally integrating with the host tisue and myelinating regenerated axons. The proposed research will develop a strategy to alter this local environment to direct differentiation of human fetal-derived neural stem cells (hCNS-SCns) into myelinating oligodendrocytes. The key factors required to promote oligodendrocyte differentiation are largely unknown and current protocols are inefficient. In Aim 1, we wil use a high-throughput array of transcription factor (TF) activity to identify these key factors within controlled microenvironments. As the output of complex intracellular signaling networks, TF activity represents the functional state of a cell (e.g., stage of differentiation). Using this novl systems biology approach, we have the ability to quantify the functional cell response to various extracellular cues and identify cues which most efficiently activate the signaling pathways that induce oligodendrocyte differentiation. Dynamic changes in TF activation will be monitored in live hCNS-SCns cultured in 3D microenvironments tuned to present combinations of defined extracellular cues simultaneously. Effects of these cues will be systematically evaluated to identify conditions that best direct oligodendrocyte differentiation. In Aim 2, hCNS-SCns will be transplanted to an in vivo model of SCI within microenvironments displaying cues found to maximize oligodendroctye differentiation. Microenvironments will be incorporated into a biomaterial platform previously developed by the Shea laboratory. These bridge scaffolds exhibit a microarchitecture that promotes axon guidance across the injury and back into host tissue. Scaffold-mediated delivery of genes encoding for neurotrophic factors further enhances axon growth. This proposal builds upon this success by adding a component to replace myelinating oligodendrocytes after SCI. In addition, Aim 2 will investigate how the stage of differentiation at the time of implantation affects the myelination capacity of hCNS- SCns. Results will significantly advance the clinical potential of cell transplantation for SCI repair. I addition, the proposed training plan will more than adequately prepare the felow for a successful career in academia. The fellow will gain expertise in human stem cell cultures, in vivo models of SCI and methods to genetically modify living cells as a tool to understand biological mechanisms. Furthermore, she will apply this knowledge to develop novel strategies for regenerative medicine. The fellow also has significant opportunities for professional development through the proposed training plan, including teaching and writing workshops, attending scientific conferences and mentoring students.